1. Technical Field
The present invention relates to a method for transforming iota-carrageenan to alpha-carrageenan by means of a novel class of 4S-iota-carrageenan sulfatase. The present invention also relates to carrageenans obtained by said conversion method.
The present invention finds application especially in the agro-food, pharmaceutical and cosmetic industries.
In the description below, the references in square brackets ([ ]) refer to the list of references presented at the end of the text.
2. State of the Art
Carrageenans are sulfated galactans extracted from the wall of marine red algae. Carrageenans are composed of a succession of D-galactosides alternately linked by alpha(1-3) and beta(1-4) bonds. These anionic polysaccharides are mainly distinguishable by the presence or otherwise of a 3,6 anhydro bridge on the galactose residue linked at the alpha(1-3) position, and by their degree of sulfation. For example, the three disaccharide repeating units—called carrabiose motif—found in the most industrially exploited carrageenans are characterized by the presence of one (kappa-carrabiose), two (iota-carrabiose) or three sulfates (lambda-carrabiose) (FIG. 1). Carrageenans may be mainly composed of a carrabiose motif, for example kappa-carrageenan from the alga Kappaphycus alvarezzi is composed of about 90% kappa-carrabiose motif and 10% iota-carrabiose. The iota-carrageenan extracted from Eucheuma denticulatum is composed of 85% iota-carrabiose units and 15% kappa-carrabiose units.
The compositions with respect to carrabiose unit may be highly variable and depend mainly on the botanic origin of the alga. The term kappa-carrageenan is used when the polysaccharide is rich in kappa-carrabiose motif and when these physicochemical properties are similar to those of kappa-carrageenan from K. alvarezzi which is often used as reference (Bixler et al., Food Hydrocolloids, 15: 619-630, 2001) [1].
A whole range of intermediate structures of hybrid kappa/iota-carrageenans has been described according to the botanic origin of the polysaccharides (FIG. 2; Bixler et al., 2001, cited above) [1]. The type of carrageenan present in the wall of algae may also be correlated with the stage of life of the algae. Indeed, in the case of Chondrus crispus, the gametophytes are rich in kappa/iota-carrageenans while the sporophytes contain mainly lambda-carrageenan. The seasons and all the environmental factors which may affect the growth of algae (illumination, temperature, salts and the like) will also have an effect on the carrageenan structure and composition. Consequently, depending on the origin and/or the procedures for extraction, a wide range of structures of the kappa-, iota- and lambda-carrageenan type may be observed.
These polysaccharides have unique rheological properties and are used as texturing agents in the agro-food, pharmaceutical and cosmetic industries. These polysaccharides have a wide range of functional properties which can be explained by their high structural diversity. The kappa- and iota-carrageenans have the property of forming ion- and thermo-dependent gels. Kappa-carrageenan will form rigid gels in the presence of potassium while iota-carrageenan forms flexible and elastic gels in the presence of calcium. The high diversity of chemical structure of carrageenans and their natural hybridity confer characteristic functional properties on each alga extract.
About 50 000 tons of carrageenans are sold yearly (Bixler and Porse, J. Appl. Phycol., 2010, online) [2]. However, the tonnage of carrageenans exploited is limited by the quantity of red alga available. Currently, two species of red alga are widely cultivated: Kappaphycus alvarezzi and Eucheuma denticulatum from which kappa- and iota-carrageenan are extracted, respectively. Numerous wild algae (not cultivated) are also collected in a large quantity because their carrageenans, of a kappa/iota-hybrid nature, exhibit highly advantageous functional properties. However, a ton of these algae is twice or even ten times more expensive than that of cultivated algae.
In addition, each industrial application corresponds to extracts of carrageenans obtained from one species or from several species of red algae. The solutions for satisfying the industrial needs in any field reside mainly in the formulation (mixture) of carrageenans (Bixler and Porse, 2010, cited above) [2].
Consequently, a biotechnological process which would make it possible to obtain hybrid carrageenans from cultivated algae would be economically highly profitable. It would have the advantage of being less dependent on the source of algae, and would open novel perspectives for the exploitation and upgrading of the biomass of red algae.
In order to control the chemical structure and, by extension, the physicochemical properties of carrageenans, the inventors therefore undertook the purification and the production of enzymes capable of modifying and correcting the structures of carrageenans. The desired modifications consist of the desulfation of carrageenans by enzymes called sulfatases which lead to the conversion of iota- to kappa- or alpha-carrageenan, or to hybrid structures of the kappa/iota- or iota/alpha-carrageenan type (FIG. 3). They thus demonstrated the existence of carrageenan sulfatases which can act directly on the polymer without preliminary action of carragenases. They succeeded in purifying, from a bacterial population Pseudoalteromonas carrageenovora, a first 4S-iota-carrageenan sulfatase belonging to the family of amidohydrolases and which converts iota- to alpha-carrageenan by specific desulfation (removal of an SO3− group) at the 4 position of iota-carrageenan (French patent application FR 09/52642) [3]. Using the same strategy as for the first 4S-iota-carrageenan sulfatase, the inventors succeeded in purifying, from Pseudoalteromonas atlantica, a second 4S-iota-carrageenan sulfatase belonging to the family of formylglycin-dependent sulfatases. They first of all succeeded in determining three peptides of said protein: NGQFDNTVIVFTSDNGGK (SEQ ID NO:1), FDQTFQVGDNTR (SEQ ID NO:2), and ETEYITDGLSR (SEQ ID NO:3), which, once compared with the TrEMBL library, demonstrated a correspondence with the Q15XH3 protein of P. atlantica T6c whose gene (Patl—0889) was labeled a sulfatase (ProteinJD ABG39415.1; Copeland et al., 2006) [4].
The 4S-iota-carrageenan sulfatases could therefore make it possible to calibrate the “hybridity” of carrageenans. Thus, any sulfatase acting on iota-carrageenan could represent a major innovation because it would make it possible to manufacture alpha- and iota/alpha-carrageenans which are very scarce in nature, and which are different in terms of peptide sequence but also in terms of biochemical properties. However, it appeared that it was possible for sulfatases acting on carrageenans not to exhibit homology with the other sulfatases known—the most widely studied sulfatases being the enzymes acting on heparine, a polysaccharide of animal origin.
A real need therefore exists for purifying enzymes for modifying the motifs of sulfation of carrageenans in order to overcome the deficiencies, disadvantages and obstacles of the prior art, in particular for a method which makes it possible to control the “hybridity” of carrageenans using said enzymes, to reduce the costs and to control the supply and the functional properties of the carrageenans thus obtained.